Haemoglobinopathies and Sickle Cell Diseases

Overview of Haemoglobinopathies

• These are disorders that involve haemoglobins, which can be qualitative, quantitative, or a combination of both.
• Qualitative disorders are exemplified by sickle cell diseases.
• Quantitative disorders include thalassaemias.
• HbE β thalassaemias represent a combined quantitative and qualitative disorder, resulting in a thalassaemic phenotype.

Characteristics of Sickle Cell Diseases

• Sickle cell diseases are a heterogeneous group of disorders characterized by red cell sickling.
• In sickle cell diseases, the haemoglobins in the red cells are mutant.
• These diseases involve qualitative defects in haemoglobin.
• The syndromes are caused by a single amino acid substitution in most cases.

Examples and Prevalence of Sickle Cell Diseases

• SS (Sickle Cell Anaemia): αβ6valαβ6val
• AS (Sickle Cell Trait): αβαβ6val
• SC: αβ6valαβ6lys
• CC: αβ6lysαβ6lys
• SD: αβ6valαβ121glu
• SS is the most common sickle cell disease, followed by AS, SC, and CC.

Pathologies in Sickle Cell Diseases

• Mutations involving the β or α genes are the most common.
• The majority of over 500 structurally different human haemoglobin variants are due to a single amino acid substitution in one or more of the globin chains.
• Genetic abnormalities are rarely present.
• There are ~70% more known β genes substitutions than α genes.
• Amino acid replacement results from a single nucleotide base substitution in the gene or messenger RNA involved in the amino acid's synthesis.

Haemoglobin Structure and Development

• All human haemoglobins consist of two α globin and two β globin chains, forming a tetrameric structure.
• Each globin chain is associated with one haem molecule and one molecule of oxygen.
• Different haemoglobins form at different stages of human development.
• At 0-8 weeks: Hb Gower I (ζ2ε2), Hb Portland (ζ2γ2), Hb Gower II (α2ε2)
• Fetal life: Hb F (α2γ2), Hb A (α2β2)
• Adult life: Hb A (α2β2) (97%), Hb A2 (α2δ2) (2.5%), Hb F (α2γ2) (0.5%)

Genetic Basis of Globin Chains

• Genes on chromosome 16 and 11 code for α-like and β-like globin chains, respectively.
• Abnormalities of genes on chromosome 11 are the most common.

History of Sickle Cell Anaemia (SS)

• James Henrick, an American physician, first described sickle cell anaemia in a West Indian student in 1910.
• In 1949, E.A. Beet and J.V. Neel determined and described its genetic transmission.
• Since Paulin et al.'s 1949 discovery of sickle cell haemoglobin, over 400 haemoglobin mutants are reported.
• In 1954, the protective effect against the lethal form of malaria of the sickle cell trait was described.

Epidemiology of Sickle Cell Anaemia

• SCA is one of the most common inherited life-threatening disorders in humans.
• Predominantly affects people of African, Indiana, and Arab ancestry.
• Over 80% of over 300,000 annual births occur in sub-Saharan Africa, particularly in Nigeria and the Democratic Republic of Congo.
• The gene frequency is highest in West African countries, with 1 in 4 to 3 (25–30%) being carriers of HbS, compared to 1/400 African Americans.
• Life expectancy is between 58 and 66 years with good healthcare; without treatment, individuals rarely reach adulthood.

Pathogenesis of Sickle Cell Anaemia (SS)

• Sickle cell anaemia involves a single base-pair point mutation of adenine (A) to thymine (T) (GAG to GTG) on the 6th codon of the β globin gene on chromosome 11.
• This mutation results in valine (hydrophobic) substituting glutamic acid (hydrophilic) at the 6th position of the β-globin chains.

Pathophysiology of Sickle Cell Anemia (SCA)

• Pathophysiology of SCA is related to abnormal haemoglobin and membrane abnormalities.

Abnormal Haemoglobin

• Haemoglobin S polymerizes only in the deoxygenated state; HbS does not polymerize when fully oxygenated.
• Sickling is the change of red blood cells from a biconcave shape to a sickle form.

Polymerization Process

• Involves the formation of liquid crystals or tactoids of HbS, which then leads to thin bundles of HbS fibres aligned along the long axis of the red cell, causing abnormal cell shape.
• Normal Hb and fully oxygenated HbS remain in solution form, but deoxygenated HbS tends to form a gel.

Factors Influencing Gel Formation

• The balance between solution and gel is influenced by O2, pH, 2,3-DPG, CO, temperature, and ionic strength.
• Factors that reduce O2 affinity (23DPG, CO, protons, high pH) cause HbS deoxygenation.
• As HbS deoxygenates, single molecules aggregate, forming homogenous nucleation.

Heterogenous Nucleation and Fibril Formation

• As deoxygenation increases, more HbS molecules aggregate to form heterogenous nucleation.
• Increased concentration of HbS and O2 supports homogenous to heterogenous nucleation.
• Hemoglobin undergoes heterogenous nucleation, forming tactoids, fibrils, and fibers within red cells.
• Fibrils and fibres run parallel to red cells, resulting in elongated shapes from elliptoid to crescent.
• The formation of a polymer starts with deoxygenation and insertion in hydrophobic pockets, leading to initiation of polymerization.

Membrane Abnormalities

• Membrane lesions in SS red cells result in:
• Loss of deformability due to parallel fibers to the long axis of the cell.
• Cytoskeletal defects. (defects with ankyrin and spectrin binding affinity)
• A reversible but balanced K+ loss and Na+ gain with no net water loss in Na+/K+ transport, but water loss in K+/Cl- transport.
• Increased adhesion of SS red cells to the vascular endothelium because of increased negative charges and secretion of adhesion molecules.

Haemolysis and Vessel Occlusion

• Haemolysis in SCA is mainly extravascular, although intravascular hemolysis can occur during the occlusion of small vessels, resulting in red cell collusions.
• Recurrent occlusion of small vessels results in repeated infarctions and necrosis in various organs.

Haplotypes of Sickle Cell Gene

• Haplotypes are groups of DNA variants inherited together from one parent and transmitted en bloc.
• Several different haplotypes of sickle cell gene mutation exist, confirming the spontaneous origin in different geographic areas.
• Common haplotypes include Cameroun, Senegal, Benin, Bantu, and Saudi-Asian.
• Clinically important haplotypes are associated with specific HbF levels; Senegal and Saudi-Asian haplotypes are associated with milder diseases due to elevated Hb F levels.

Inheritance Pattern

• Inheritance follows a simple autosomal recessive pattern.

Sickle Cell Habitus Traits

• Slender and tall patients, though not necessarily above-average height, frequently indicate sickle cell habitus.
• Linear body shape with narrow shoulders and hips.
• Dolichocephalic or scaphocephalic head shape can manifest in some cases.
• A short neck also can be another indicator.
• Accentuated upper dorsal kyphotic curvature and exaggerated lower lumbar lordotic curvature of the spine.
• Thin and long arms and legs with a short trunk.
• Appearance: hoop-chested.
• Long, narrow hands and fingers, feet and toes, described as "spider hands".
• Presence of hypogonadism signs: (genital hypoplasia or atrophy, hypotrichosis, high-pitched voice)
• In children, signs range from slight to significantly unusual, including thin limbs, "hoop chest," and protruding abdomen.

Anaemia

• Triggered by marked reduction in RBC lifespan because of an increased rate of destruction.
• May get worse by lack of folic acid.
• Patients tend to have a lower baseline PCV, known as a stable PCV, which ranges between 18-22%.

Sickle Cell Crisis

• Defined as sudden onset of new syndrome(s) in individuals, requiring immediate medical attention.
• Defined as abrupt and hazardous changes in chronic haemolysis.
• Manifests in three forms: Vaso-occlusive crisis, acute sequestration crisis, and aplastic crisis.

Vaso-occlusive Crisis

• Comprises painful bone crisis, acute chest syndrome, acute abdominal pains, and acute CNS events, for example CVA in about 7% of patients.
• Attributed to the blockage of small vessels in vital organs.

Acute Sequestration Crisis

• Acute and sudden pooling of red cells in the spleen causing possible circulatory collapse.
• Major cause of death in children under age 2 years.

Aplastic Crisis

• SS patients have compensated anaemia by increasing bone marrow output 6-9 folds.
• When the bone marrow or specifically erythroid precursors are suppressed either by bacteria or viral infections commonly viral parvovirus B19 there is a sudden and dangerous drop in PCV.

Infections

• Common death cause in SS patients.
• Pneumococcal sepsis carries 40 times the risk of normal, while H. influenzae is 2-4 times normal.
• Caused by host immunity defects (splenic dysfunction, abnormal neutrophil chemotaxis).
• Functional asplenia during the first year of life, which leads to an inability of the spleen produce antibodies, and to clear particles from the intravascular space.
• Abnormal neutrophil chemotaxis also contributes to higher infection risks.

Chronic Organ Damages

• Cardiovascular System (CVS): systolic ejection murmur, diastolic murmur, cardiomegaly (most patients), left ventricular hypertrophy (50% of patients)
• Myocardial infarction is rare.
• Renal: hyposthenuria, haematuria, uraemia, nephrotic syndrome
• Hepatobiliary System: cholelithiasis (gallstones), hepatic infarction, transfusion-related hepatitis
• Eyes: tortuosity and sacculation of conjunctival vessels, proliferative and non-proliferative retinopathies
• Skin: leg ulcers (adolescence and adulthood, media surface of the lower tibia or posterior to the medial malleolus)
• Ears: sensorineural hearing loss
• Skeleton: Frontal bossing and hand and foot syndrome, skeletal changes (increased bone marrow activity/repeated bone marrow infarction)
• Chronic back pain.
• Lungs: pneumonia and pulmonary infarction

Growth and Development

• Initially normal birth weight.
• Subsequently, delayed growth and development, followed by reaching normal height.
• Weight gain is usually not fully achieved.

Diagnosis of Sickle Cell Anaemia

• Incorporates history, physical examination, and laboratory findings.

Laboratory Findings for Diagnosis

• FBC: anaemia, low PCV, increased MCHC
• Increased WBC and platelet counts, reactive but not too high.
• Blood film: normocytic normochromic RBC, sickle RBC.
• Sickling test: Na metabisulphite.
• Haemoglobin electrophoresis to confirm diagnosis.

Managing Sickle Cell Anaemia

• Management spans preventive, supportive, and definitive measures.
• Preventive measures: prevent dehydration and maintain adequate fluid intake in hot weather; to avoid infected leg ulcers by using general hygiene measures; avoid hypoxia with air travel, high altitudes, or anaesthesia.
• Also includes preventing exposure to cold situations and prompt treatment of infections.

Supportive Measures

Anaemia

• Red cell transfusion only under special circumstances. Raise packed cells to stable state level or slightly above it. As a result a unit of blood raises PCV by 3% or raises Haemoglobin concentration by 1g/dl.
• Exchange transfusion may be indicated at times of increased risk e.g during pregnancy, surgery.

Painful Crisis

• Rehydration: Administering hypotonic fluids to promote water entry into red cells.
• Adequate pain relief: Strong analgesics such as pethidine, DF118, and/or morphine.

Infections

• Prophylactic penicillin or antibiotics at first sign of infections.
• Surgery: Major surgery increases risk; HbS level should be reduced to less than 20% prior to major surgery by exchange blood transfusion or repeated transfusion with AA blood.
• Pregnancy: Carries a higher rate of abortion and stillbirths. Better general ANC and routine drug intake are better for the patient.

Definitive Measures

• Splenectomy for recurrent acute sequestration crisis.
• Anti-sickling drugs (use cautiously): cyanate, 5 azacytidine (stimulate production of γ globin).
• Hydroxyurea is commonly used to stimulate production of Hb F.
• Bone marrow and gene therapy

Pre-Natal Diagnosis for Haemoglobinopathies

• Commonest single gene disorder that constitutes public health issues worldwide.

Fetal Blood Sampling:

• A prenatal blood test may be done late in the 2nd trimester. Furthermore, they is relatively safe and reliable ways to carry out the process.
• Electrophoresis of the sample and globin chain synthesis are measured.
• Therapeutic abortion can be a thing.
• Another disadvantage can be having to wait a long duration for mother, this occurs as we carryout the process during late 2nd trimester.

DNA Analysis

• A analysis that allows you to obtain Fetal DNA from amniotic fluid, as well as chorionic villi samples.
• The process occurs quite early.
• However, at the start of termination process one can see that the results are unfavourable.
• It is to be known that fetal loss may be a big disadvantage.